Understanding the influence of bone-metastatic prostate cancer and mesenchymal stromal cells on γδ T cells, in the bone microenvironment.

虽然免疫疗法在治疗其他癌症方面取得了很大进展，但阉割耐药前列腺癌 (CRPC)在很大程度上仍然没有反应，突显出需要新的办法。一种这样的方法依赖于 转基因自体T细胞表达嵌合抗原受体(CAR)的研究 识别特定的肿瘤相关抗原。前列腺干细胞抗原(PSCA)广泛表达于 前列腺癌和我们之前已经开发了一种靶向PSCA的汽车，具有很强的体内疗效。至 靶向骨转移性CRPC(MCRPC)，我们建议操纵T细胞的特定亚群，称为γδ 可以通过使用双膦酸盐(如唑来膦(ZOL))的全身治疗而驱动到骨骼 临床上用于限制男性骨质疏松症患者因癌症引起的骨病。重要的是，ZOL治疗 诱导γδT细胞检测到的磷抗原在肿瘤细胞中积聚。我们的预赛 研究结果表明，ZOL可以促进γδT细胞向骨骼的归巢，在那里他们可以通过 CAR和内源性T细胞受体(TCR)识别。γδCAR-T治疗，存在或不存在 ZOL，可以缓解癌症引起的骨骼退化。此外，我们还发现，可溶性因子由 骨髓间充质基质细胞可增强γδCART细胞的细胞毒活性。 最后，我们发现CAR结构和共刺激基团的选择会影响表型和 功能不同的αβ和γδT细胞，需要针对γδT细胞进行优化的CARS设计。基于这些 初步发现我们假设γδCAR-T细胞的归巢和细胞毒活性对 骨转移CRPC的治疗可以通过遗传学、药理学和 微环境方法。我们将通过以下方法验证我们的假设：1)定义最优γδCAR-T设计 这将显著增强CRPC的细胞毒性。我们将测试选择替代方案的生物学影响 CAR跨膜和共刺激结构域，特别关注它们调节细胞周期的能力 细胞因子受体的表达。我们还将剖析可以调控γδCAR的特定信号通路- T细胞持久性。最后，我们将识别不同类型汽车所触发的分子信号通路 共刺激结构域。2)确定ZOL是否能促进γδCAR-T募集和抗骨MCRPC活性 活着。我们将使用异种移植和pdx模型来研究mCRPC的生物利用度和治疗效果。 γδCAR-T+ZOL的有效性；以及绘制磷酸抗原聚集位置的免疫活性模型。 3)解剖γδCAR-T对骨微环境的交互作用。我们将分析 γδCAR-T+ZOL治疗对骨组织结构和生理的影响 γδCAR-T细胞在体内的表现。根据预期结果，表征具体的 骨/肿瘤微环境的特性将揭示新的见解，从而提供强有力的理论基础 为翻译免疫疗法量身定做，以消除目前无法治愈的骨mCRPC。

While immunotherapies have made strides in the treatment of other cancers, castrate resistant prostate cancer (CRPC) remains largely unresponsive, underscoring the need for novel approaches. One such approach relies on the administration of autologous T cells genetically modified to express a chimeric antigen receptor (CAR) that recognize specific tumor-associated antigens. Prostate Stem Cell Antigen (PSCA) is widely expressed in prostate cancer and we have previously developed a CAR targeting PSCA that has potent in vivo efficacy. To target bone metastatic CRPC (mCRPC), we propose to manipulate a specific subset of T cells, called γδ that can be driven to the skeleton via systemic treatment with bisphosphonates such as zoledronate (ZOL) that is clinically used to limit cancer-induced bone disease in men with bone mCRPC. Importantly, ZOL treatment induces accumulation of phosphoantigens in tumor cells, which are detected by γδ T cells. Our preliminary findings show that ZOL can enhance γδ T-cells' homing to bone where they can prevent cancer growth via CAR and via endogenous T-cell receptor (TCR) recognition. γδ CAR-T treatment, in presence or absence of ZOL, can mitigate cancer-induced bone deterioration. Moreover, we found that soluble factors secreted by bone marrow derived mesenchymal stromal cells (MSC) can increase the cytotoxic potential of γδ CART cells. Finally, we found that the choice of CAR structural and costimulatory moieties affects the phenotype and fuction αβ and γδ T cells differentially, requiring the design of CARs optimized for γδ T cells. Based on these preliminary findings we hypothesize that the homing and cytotoxic activity of γδ CAR-T cells for the treatment of bone metastatic CRPC can be greatly enhanced through genetic, pharmacological, and microenvironmental approaches. We will test our hypothesis by; 1) Defining the optimum γδ CAR-T design that will significantly enhance CRPC cytotoxicity. We will test the biological implications of choosing alternative CAR transmembrane and costimulatory domains, with a specific focus on their ability to modulate the expression of cytokine receptors. We will also dissect the specific signaling pathways that can govern γδ CAR- T cell persistence. Finally, we will identify the molecular signaling pathways triggered by CARs with different costimulatory domains. 2) Determining if ZOL can drive γδ CAR-T recruitment and anti-bone mCRPC activity in vivo. We will use xenograft and PDX models of bone mCRPC to characterize the bioavailability and therapeutic efficacy of γδ CAR-T + ZOL; and an immunocompetent model to map sites of phosphoantigen accumulation. 3) Dissecting the reciprocal effects of γδ CAR-T on the bone mCRPC microenvironment. We will analyze the impact of γδ CAR-T + ZOL treatment on the structure and physiology of the bone, and the effects of MSC on the performance of γδ CAR-T cells in vivo. Based on the anticipated results, characterizing the specific properties of the bone/tumor microenvironment will reveal novel insights thereby providing a strong rationale for the translation of immunotherapies tailored to eliminate currently incurable bone mCRPC.